PROJECT SUMMARY/ABSTRACT It is well known that oxidative stress contributes to the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). However, the exact mechanism whereby changes in the oxidative environment contribute to disease has remained a "black box", and methods to detect specific oxidative modifications in target proteins have been limited. The adverse molecular actions of oxidants have largely been attributed to their role in causing damage. Yet, the concept that oxidants are molecules with regulatory functions important in normal cell and tissue homeostasis has also emerged. The latter signaling function of oxidants has been attributed to cysteine oxidations, such as S-nitrosylation (PSNO), and S-glutathionylation (PSSG). Recent work from our laboratory has determined that PSNO and PSSG are important biochemical events that limit the activation of the pro-inflammatory and pro-survival transcription factor, nuclear factor kappa B (NF-kB) in lung epithelial cells. Importantly, activation of NF-kB has been observed in patients with COPD and is believed to contribute the inflammatory process observed in these patients. To this date, information about changes in the homeostasis of PSNO and PSSG in lungs of patients with COPD, and the interplay between these events, remains largely absent, due to the difficulties to detect these oxidative modifications in situ in tissues. Our laboratory has recently developed two new assays that, for the first time, enable the detection of PSNO and PSSG in paraffin-embedded sections in situ. These recent observations and novel approaches lead us to hypothesize that the homeostasis of PSNO and PSSG is affected in lung tissues from patients with COPD, and that these changes correlate with regional patterns of NF-kB activation. In Specific Aim #1, we will determine the extent of PSNO and PSSG in lung biopsies of patients with COPD. We will utilize the recently developed chemical and catalytic cysteine derivatization strategies to monitor patterns in PSNO and PSSG in paraffin-embedded biopsy specimens from patients with varying severities of COPD using confocal laser scanning microscopy. We will also evaluate normal tissue derived from patients undergoing surgery for lung cancer resection. We will also further validate our assays and implement internal standards in order to improve their diagnostic utility. We will measure expression of glutaredoxin-1 (Grx1) and S-nitrosoglutathione (GSNO) reductase, as these enzymes represent the major regulators of PSSG and PSNO content, respectively. In Specific Aim #2 we will determine whether changes in PSNO and PSSG of NF-kB pathway members can be detected in patients with COPD. Completion of these Specific Aims represents an important validation of the utility of the new assays to detect PSNO and PSSG in tissues in situ. Finalization of this project will yield new insights into the extent to which various cysteine oxidations occur in patients with COPD, and provide insights into their relationship with the status of activation of NF-kB. (End of Abstract) PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE it is well known that oxidants contribute to damage of lungs of patients with Chronic Obstructive Pulmonary Disease (COPD). However, the exact cellular effects of oxidants have remained unclear which is problematic, because is does not enable investigators to determine whether antioxidant therapies were adequately effective. This laboratory has developed new assays which allow investigators for the first time to study the subtle action of oxidants in tissue slides prepared from lungs. This grant proposal therefore has as its major goal to test the utility of these two new assays to detect oxidative changes in normal lung as well as in lung tissue prepared from patients with increasing severities of COPD. If successful, these new assays may have diagnostic use in testing the extent of oxidative stress in patients with lung disease, and also in determining the effectiveness of antioxidant therapies. Completion of this grant proposal may also offer new insights into the mechanisms whereby oxidants regulate the severity of inflammation.